[Sirolimus inhibits the expression of type I collagen and fibronectin in cultured renal cortical myofibroblasts].
OBJECTIVE
To investigate the anti-fibrotic effect of sirolimus (rapamycin) at the cell level.


METHODS
The primary cultured rat renal cortical myofibroblasts were divided into two groups, control group and sirolimus 40 mg/L group at each time point. The protein levels of alpha-SMA, Col-I, fibronectin(FN) were analyzed by Western blot in both the whole cell lysates and supernatant culture media 12 h , 24 h and 48 h after incubation, respectively. Real-time quantitative PCR was carried out to measure the levels of procollagen-I mRNA 1 h, 2 h, 4 h, and 6 h after cell incubation. The activities of gelatinase MMP-2 and MMP-9 in the supernatant from the cultured cell media were assayed by gelatin zymography.


RESULTS
(1) Sirolimus had no effect on the expression of alpha-SMA of myofibroblasts at different time points. (2) The expression of Col-I in the whole cell lysates both reduced at the end of 24 h and 48 h in sirolimus group significantly [(0.58+/-0.05) and (0.63+/-0.18), P < 0.05] compared with control group at each time point, respectively. (3) The levels of procollagen-I mRNA reduced significantly at the end of 1 h and 2 h compared with control group at each time point [(0.38+/-0.05) and (0.55+/-0.16), P < 0.05], but increased to basic level at the end of 4 h. (4) The myofibroblasts had basic expression of Col-I early at the end of 12 h, its expression in supernatant culture medium reduced obviously both at 24 h and 48 h in sirolimus group compared with control group of each time point [(0.59+/-0.25) and (0.52+/-0.21), P < 0.05]. (5) The expression of FN in the whole cell lysates had the same trend as that in supernatant culture medium, which reduced obviously at the end of 24 h in sirolimus group compared with control group at each time point [(0.44+/-0.09) and (0.40+/-0.15), P < 0.05], but the inhibitive effect of sirolimus on FN disappeared at the end of 48 h. (6) The activities of MMP-2 and MMP-9 in the supernatant culture media were not significantly changed along with the experimental time points.


CONCLUSIONS
Sirolimus may exert its anti-fibrotic effect through the inhibition of the expression of Col-I and/or FN in cultured renal cortical myofibroblasts.
Zhang
Lan
L
Department of Nephrology, Peking University First Hospital; Institute of Nephrology, Peking University, Beijing 100034, China.
Huang
Hai-chang
HC
Li
Jing-zi
JZ
Liu
Ying
Y
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D005355:Fibrosis; D007672:Kidney Cortex / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020123:Sirolimus / Q000494:pharmacology*
D000199:Actins; D024042:Collagen Type I; D005353:Fibronectins; D012333:RNA, Messenger; D020123:Sirolimus